This study was conducted by the IVF Center at Kocaeli University, Turkey. It aimed to test the effect of biochemical markers in follicular fluid, such as nitrous oxide (NO), reduced glutathione (GSH) and malondialdehyde (MDA) on the outcome of in vitro fertilisation. The researchers selected 62 infertile women, all of whom were aged between 25 and 32, were non-smokers, had no systemic diseases, and were suffering from unexplained infertility with no signs of hormonal or ovulatory issues.

Ovulation was first induced in the women using long and short agonists along with a microdose flare-up, after which an oocyte was collected. This was followed by intracytoplasmic sperm injection (ICSI) as the preferred method of fertilisation, then the transfer of the embryo took place. To collect the FF samples, the dominant follicles were chosen and samples were centrifuged, supernatants were removed and stored.

To measure MDA levels, MDA was mixed with thiobarbaturic acid (TBA) which reacted to form a red compound. This mixture was incubated, cooled and assayed to read the absorbance of the butanol phase, and results were expressed in micromoles of MDA. Similarly, levels of GSH were measured in micromoles/L after being supplemented with metaphosphoric acid and assayed. However, NO levels were measured indirectly by measuring the nitrate and nitrite sample concentrations, deproteinising the sample, then measuring absorbance at 545nm using Griess reagent, expressing NO levels in nanomoles/L.

The embryos were then graded in terms of the size of the blastomere and degree of fragmentation, into Grades A-C, A being an even blastomere with <10% fragmentation. A single Grade A embryo was then transferred into each woman on Day 3 of the trial, after which they were categorised into 2 groups based on blood concentration of human chorionic gonadotropin; successful pregnancy (Group 1) and unsuccessful pregnancy (Group 2).

Finally, to analyse these results, the researchers used the women’s pregnancy status following IVF as the primary outcome measure. Statistic analysis was carried out according to non-parametric Mann-Whitney U test. The study found that the successful pregnancy group had significantly lower levels of fluid NO and significantly higher levels of fluid MDA than the unsuccessful pregnancy group. In analysing the correlation between IVF parameters and oxidative stress, the findings showed a positive weak correlation of MDA with fertilisation rate and the number of Grade A embryos. Also, ROC curve analysis implicated MDA as a highly sensitive predictor of pregnancy. Due to this significant difference in MDA levels between groups 1 and 2, the study concluded that MDA was the most suitable indicator of IVF success out of the 3 biochemical markers chosen for analysis.

The objective of this study was to research the possible association between levels of plasma D-dimer, a haemostatic marker, and the success or failure of pregnancy in women undergoing IVF. The researchers selected 105 infertile women from the Ortona General Hospital’s Assisted Reproduction Unit who were undergoing IVF, indicated by factors such as tubal factor, endometriosis, male factor, anovulation and unexplained infertility.

The participants underwent ovarian stimulation by receiving daily subcutaneous injections of recombinant FSH (follicle stimulating hormone) with doses varying based on basal FSH level, the age of the woman and antral follicle count. This stimulation was begun upon complete pituitary suppression on the 3rd day of the menstrual cycle, and throughout the stimulation, hormonal fluctuations of luteinising hormone (LH) were controlled by injecting agonists and antagonists of GnRH.

Following the collection of oocytes, intracytoplasmic sperm injection (ICSI) was used to perform IVF and an embryo transfer was carried out, 14 days after which a pregnancy test was taken. The presence of a gestational sac with a fetal heartbeat after 7 weeks of gestation was the criterion for a clinical pregnancy.This ovarian stimulation protocol was followed by a venous blood sample to test D-dimer concentrations, where a latex quantitative assay was used with 200ng/mL being the threshold level for an abnormal D-dimer level. These assay levels were statistically analysed using a Mann-Whitney U-test and T-test.

The results of the study indicated significantly higher levels of circulating D-dimer in women with a failed pregnancy following IVF in comparison to those with a clinical or successful pregnancy. This difference was statistically valid even when taking age and vascular risk factors into account. It was found that women with concentrations of D-dimer above the threshold had a more dismal pregnancy outcome, and that D-dimer levels increased after the one-week administration of GnRH. Overall, only 38% of the participants had achieved a clinical pregnancy and the study concluded that high D-dimer concentrations are implicated in a higher risk of a failed pregnancy following IVF. This was consistent with previous postulations that a possible mechanism for failure is unsuccessful implantation and placentation, owing to a hypercoagulable vascular state, leading to increased risk of thrombolic events in maternal vessels to the placenta.

--Mark Hill - Both these papers are recent and present interesting findings. It would have helped you summary in the second paper if you had described what plasma D-dimer was. Your summaries are both correct and concise (5/5).

Lab 2

Fertilisation of medusa eggs by spermatozoids in vitro in sea water[1]

--Mark Hill (talk) 16:28, 21 August 2014 (EST) This is all correct, I have fixed the reference link below. Please in future use a shorter image title and do not use .jpg in the image description above (I have removed for you). (4/5)

--[[User:Z8600021|Mark Hill] These references are appropriate, you should have included some descriptions, even a single line, with the reference (4/5).

Lab 4

Summary of research article

This study by Chang et al. observed the effect of varying the intratracheal transplantation time of umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) on the attenuation of hyperoxic lung injury in neonatal rats. It was concluded that the optimal time window for stem cell transplantation was narrow; efficient only during the early but not late phases of inflammation. The findings were in line with previous research that the anti-inflammatory properties of UCB-derived MSCs play a crucial therapeutic role in the alleviation of bronchopulmonary dysplasia (BPD), through reducing hypoxia-induced injuries including increased apoptosis and impaired alveolarisation.

Tissue sampling and analysis of lung histopathology found that the Hypoxia Control (HC) group showed fewer, larger and more abnormally-sized alveoli compared to the Normoxia Control (NC) group. However, upon transplantation of UCB-derived MSCs, these alveolar impairments and changes in morphology were attenuated, especially when added on post-natal day 3 (HT3) than on post-natal day 10 (HT10).

The study’s comparison of the levels of pro-inflammatory cytokines such as IL-6α, IL-6β and TNF-α before and after the addition of the UCB-derived MSCs found that such molecular markers were significantly lower after HT3 transplantation than HT10 transplantation. These levels were much lower than the initial starting levels exhibited by the NC group, indicating the therapeutic effect of the cord cells. Furthermore, the hypoxia-induced reduction in VEGF and HGF levels and increase in lung collagen levels were both attenuated by the addition of the MSCs.

These findings led to the conclusion that the therapeutic efficacy of UCB-derived MSCs on treating BPD is indeed time-dependent; having potent effects in the early inflammatory process, which is then reduced in later stages. The study also tested any potential synergistic effects of combined early and late MSCs intratracheal transplantation, but none were found.

The 3 developmental vascular shunts in the fetal circulation that close postnatally are:

a) Ductus arteriosus (Ductus Botalli): Connects the pulmonary artery to the proximal descending part of the aortic arch and prevents the output of the right ventricle from entering the unexpanded fetal lung, which is fluid-filled and non-functioning. This shunt transfers medium oxygen saturated blood and becomes the ligamentum arteriosum after closing at birth.

b) Ductus venosus: Shunts blood from the left umbilical vein to the inferior vena cava. This carries well-oxygenated blood and allows placental blood to bypass the liver ultimately to the fetal brain.

c) Foramen ovale (Foramen Botalli): Shunts blood that is highly saturated with oxygen from the right atrium to the left atrium and becomes the fossa ovalis after closing at birth.

Lab 5

Bronchogenic Cysts

Bronchogenic cysts (BCs) are an unusual and rare congenital abnormality of the bronchial tree; a generally benign type of malformation of the bronchopulmonary foregut. The classification of these cysts is into; those occurring in the mediastinum (65-90%), which have equal prevalence between the sexes, or those of intrapulmonary origin (lung or pleura) which display a slight male predominance [1] Other sites such as intradiaphragmatic and retroperitoneal are rare, and consequently require close attention.

These lesions are characteristically unilocular, mucus field cysts which arise from the posterior walls of airway membranes during embryonic development. They currently account for 20-30% of congenital cystic bronchopulmonary foregut abnormalities and for only 5-10% of paediatric mediastinal masses. [1] Although the exact cause of bronchogenic cysts is yet to be established, current research postulates that that their developmental cause is the abnormal budding of the tracheal diverticulum and proximal bronchial structures during embryogenesis in weeks 4-6. Bronchogenic cysts stem from abnormal buds from the tracheobronchial tree or primitive esophagus which fail to extend to the site of alveolar differentiation. Generally, early separation causes the system to move into the mediastinum, however it is when this separation occurs late that a BC forms [1]. Histologically, these abnormal bronchogenic cysts are lined by pseudostratified, ciliated, columnar or cuboidal secretory respiratory epithelium and their walls comprise typical structural components of the airways such as smooth muscle, cartilage and mucinous glands. Cysts are typically not filled with air as they lack communication with the bronchial tree, but contain fluid comprising blood, proteinaceous fluid and calcium oxalate, resulting in a solid lesion manifesting on radiographic imaging such as MRI and CT scans [2]

BCs are considered to be the most common form of foregut duplication cysts and are a congenital anomaly as they are asymptomatic and rarely arise in infants with diagnosis usually occurring post-natally. The clinical presentation of cysts is usually either asymptomatic or through airway obstruction causing respiratory distress or cystic infection causing compression of central lung parenchyma [3]

--Mark Hill This was an interesting abnormality that you have reviewed here. Note that the cause is still speculated and there is not a good animal model for this condition. (5/5)

Lab 7

Research article- Pax6 is essential for establishing ventral-dorsal cell boundaries in pituitary gland development

This study by Kioussi et al. investigated the role of the transcription factor Pax6 in creating a dorsal-ventral boundary between cell types of the developing pituitary gland. The researchers considered the physical location of the six cell types in the pituitary that originate from a common primordium- corticotropes, thyrotropes, menalotropes, gonadotropes, somatotropes and lactotropes. They specifically focused on the regulation of cell differentiation by Pax6 and the effects of its absence.

Following analysis of mutant mice embryos with a Small eye (Sey) mutation and Pax6 gene deletion, the study found that Pax6 plays a significant role as an early dorsal marker of the pituitary gland’s development. They found that Pax6 transcripts were expressed and present in the nascent Rathke’s pouch, however excluded from the ventral section of the Sonic hedgehog (Shh) pouch, leading to the formation of a ventral zone between cells expressing Pax6 and those expressing Shh. A dorsal-ventral gradient for cell differentiation was evident and Pax6 was seen to be excluded from the rostral zone of the pituitary. These two particular findings suggested that Pax6 may be an essential element of the ‘transcriptional apparatus’ responsible for determining the position of the six pituitary cell lineages.

Furthermore, the research concluded that the absence of Pax6 leads to a decrease in dorsal cell types from the Pit-1 lineage (somatotropes and lactotropes) and an increase in cell types with ventral phenotypes such as thyrotropes. This was confirmed by discovering that the expression patterns of four molecular ventral markers such as GATA2 were uniformly dorsalised, leading to further increase in the population of thyrotropes.

Hence, these results implicated that Pax6 functions as a transcriptional regulator in opposition to ventral signalling molecules, thus clearly delineating the developing dorsal cells from ventral cells in the differentiation stage of pituitary gland development. It has a key regulatory role in the formation of a sharp dorsal-ventral cell type margin through the inhibition of Shh ventral signals.

The cells and their layers that contribute to tooth development through odontogenesis (from week 6 of development) include:

Odontoblasts: Mesenchymal cells of neural crest origin that produce predentin, which calcifies forming dentin in the process of dentinogenesis. Enamel epithelium causes odontoblast differentiation and these cells contribute to the outer dental pulp

Ameloblasts: Derived from ectodermal cells of oral epithelium tissue. They differentiate from preameloblasts, activated by ectomesenchymal cells and produce enamel proteins such as amelogenin and enamelin to form enamel, the outer covering of the tooth’s crown.

Peridontal ligament: Composed of connective tissue (bundles of collagen fibres), which secures the root of the tooth in the alveolar socket so it is not displaced. It surrounds the cementum of the tooth root.

--Mark Hill Note that this is a fairly "old" article and there is current information about Pax6 that clarifies its role more in pituitary development. (5/5)

Lab 8

Ovary development

Initially, the genital systems of both sexes are morphologically similar and sexual development hence begins as an indifferent stage with an indifferent (‘bipotential’) gonad later forming either the testis or ovary. Both gonads have contributions from; mesothelium of the posterior abdominal wall cavity, mesenchyme beneath this and primordial germ cells (PGCs) that later differentiate to form sex cells.

In Week 5 of development, a thickening of mesothelium forms on the medial portion of the primitive kidney (the mesonephros). The formation of the gonadal ridge results from the proliferation of the mesothelium and mesenchymal tissue beneath it, as a bulge on the medial mesonephros. Projection of finger-like gonadal (epithelial) cords into the mesenchyme follows, segmenting the indifferent gonad into an internal medulla (covered by germinal epithelium) and external cortex. For embryos with a sex chromosome complex of XX, the medulla regresses while the cortex of the indifferent gonad differentiates to form the ovary.

PGCs reside among dorsal endodermal cells of the umbilical vesicle whose dorsal part is incorporated into the embryo during folding. They are the first cell type migrating through the primitive streak in gastrulation (3rd week) and then reside at the junctional region of the hindgut yolk sac. Following this, there is migration of the PGCs to the gonadal ridge along the hindgut’s dorsal mesentery, regulated by BMP-4, fragilis and stella genes. In week 6, these germ cells are incorporated into the gonadal cords and are called oogonia, having entered underlying mesenchyme.

In female embryos, ovary development is a slow process, with the ovary not histologically identifiable until week 10. Ovary development relies on the genes of the XX chromosome along with an autosomal gene. Gonadal cords form a basic rete ovarii (network of canals) by extension into the internal medulla, although the cords are not notable in the embryonic ovary. The rete ovarii and gonadal cords usually degrade and cease to exist.

--Mark Hill Very good I only required the embryonic component of ovary development described. I prefer you not to use the textbook as your source. (4/5)

Lab 9

Group 1- Respiratory

Overall, the project at this stage consists of a good integration of text, images and references throughout. The introduction is well-written and gives an overview of the two parts of the respiratory system (conducting and respiratory zones). I think it is a good idea that your group has split this up and explained both parts separately as it helps to orient an unknowing reader, especially as the gross anatomical structures are also described (e.g. trachea, larynx, bronchi). However, in-text referencing is needed in this introductory segment to provide the reader with the source of all information, exactly where it appears. You could refer to Dr Hill’s instructions on how to do this if needed, or see another group’s page on Edit mode. Also, the images used in the introduction should have a small caption beneath them, otherwise it is hard to tell what the images show exactly and how this may be relevant to the complementary text.

In terms of heading and subheading organisation, I like how you have split the content up into 5 main areas of introduction, lung development stages, current research models/findings, historic findings and abnormalities. This makes the page easy to navigate and the subheadings under each section are relevant. The use of a table in the ‘lung development stages’ section is very well done and appropriate, as it segments the information into a clean, readable format that a student could simply refer to if they were learning from scratch. The information in the table is succinct and provides all the main points. The only improvement here I would suggest, is aligning the content to the left, as it may seem more pleasing to the eye to have even spacing rather than centre alignment. Also, the ‘references’ have been placed as subheading 2.1, whereas the other ‘reference’ sections have not been given a separate subheading, so I would consider making this consistent throughout the project page.

The section on current research models and findings is concise and informative, with good use of numbering to make the information easier to read rather than having long and chunky paragraphs. Although a minor detail, there is one part that says “a study conducted last year”. Since these Wiki pages will be left online, it is important to specify the exact year here, and provide an in-text reference to the study mentioned so a reader can easily locate it. I like the use of dot points in this section, making it look appealing, however the image used should also include a caption, as should the others on the page. Be careful of copyright infringement regarding image use, as there appears to be a file with a ‘Permission Error’ in this section, which may need to be manually removed.

The ‘historic findings’ section was also well-done, especially because it used dot points to segment the information and show the exact years of each discovery. Once again though, the image requires a caption and the references for this section seem to be split into 2 parts; one list from 1-14 then another from 1-4. I think the list from 1-4 needs a subheading to show how those sources are different to the ones above it, otherwise both lists need to be integrated into one.There are also some parts that have coding showing </ol> and </span> </li> which just need to be removed with editing.

Lastly, the section on abnormalities is also of a high standard as each abnormality begins with a brief description then goes into details by using dot points. There is good use of in-text referencing followed by a reference list which is correctly formatted too. The image included has a caption which is good, as other sections lack this, however I would consider adding more images to make this part more visually appealing and engaging to the reader. It is evident that a lot of work has been done on this page as each section is detailed and referenced well, with relevant information.

Group 2- Renal

The introduction provided by this project is very good and includes in-text citations. Not only does it introduce the renal system’s components but also discusses its development briefly into the embryonic and fetal stages, focusing more on fetal. Also, by having the references as one long list at the very end of the page, this gives the project a clean and tidy look, which some of the others lack. I thought this was a great idea and very orderly.

While some sections are full of information, others are scarce or empty, such as the ‘Historical findings’ section. Some information on the development of knowledge on the renal system throughout history should be included here, maybe making use of dot points detailing specific year dates. The ‘Developmental Timeline’ provides a good overview of the system’s development, although weeks 3-5 may not be necessary as these are during the embryonic period and the focus here is on fetal development. However, it does provide an overall context which is good. This information may also be effectively translated into a table format for easier readability.

The section on ‘current research models’ was nicely written, with solid analysis of 2 research articles. Using any more articles to that level of depth may be too much information, so this is a good balance. It was very good to see the text actually explaining the accompanying image, which was labelled with a caption too. The introduction to the use of animal models and why these are important was effective also.

I particularly thought the sub-sectioning of the page into the main organs of the renal system was a smart idea rather than having the entire system as a clump of information as this way, it is easier to navigate through the information. The ‘kidney’ section was of a very high standard as the information was relevant and nicely split into different processes of fetal development such as nephrogenesis and renin production. It is very helpful as a reader to have explanations of the images used, making the page more interactive and useful. However, this is a file with a ‘Permission error’ present which would need removal due to copyright infringement; this should be sorted out before the project is due.

The following three sections on the urethra, ureter and bladder were also well-written, referenced correctly with in-text citations and the images used were relevant to the text. However, captioning the image in the urethra section would be good to give the reader knowledge of exactly what it shows. The ‘abnormalities’ section was again, well -researched and full of information, however it seems a little cluttered as lengthy references are placed under the text. Integrating these into the overall reference list at the end of the page would look clearer.

Overall, this project has been well done and there is evidence of consistency throughout the section formatting, suggesting the group members have been communicating between each other, which is good to see. Some improvements I would suggest are the use of hand-drawn images to make it easier for a student to learn off the project, and using tables to summarise some information e.g. timeline.

Group 3- Gastrointestinal

The introduction part of this project provided a good overview of the gastrointestinal tract and its components, also mentioning briefly the changes that occur in the fetal period. However, care must be taken to not capitalise words that are not needed e.g. Foregut, Midgut, Appendix etc. Although the information in the ‘timeline’ section is relevant, its formatting needs a bit of review as there are inconsistencies between the foregut, midgut and hindgut parts. It also seems that the in-text citations have just been arbitrarily placed in chronological order, with each line having a new reference. Also, this information may be better presented in table format to improve readability. Some simple editing may be needed to fix this.

The section on ‘Recent findings’ has some good information, however the reference needs to be properly cited and maybe a couple more articles would help give this part some substance. I thought it was effective to have the GIT split into the foregut, midgut and hindgut and then detail the fetal development under those titles. This gave the page a good structure. However, adding some images, both from online and student-drawn to the foregut and hindgut sections would help improve the aesthetics of the page. The hand-drawn images on the midgut section are effective learning tools to a student, but maybe could be drawn a little neater with darker colours as the blue labelling is difficult to read. They should also be captioned. The use of a table to show midgut herniation of fetuses was a good tool as it makes the information easier to read also.

Lastly, the section on abnormalities was well-detailed and I liked that the deformities were split into a definition and cause. An image of gastrochisis may be helpful for a reader to visualise the condition though. The in-text citations in this part were properly done and a long list of references being at the end of the page made the project look neat overall. Areas of improvement may be some simple formatting changes and evening out the information across sections, however a solid project so far.

Group 4- Genital

Overall, it is evident that a lot of work has been completed on this project as each section has a decent amount of information and there are images throughout the page. However, the addition of an ‘introduction’ section would help to orient the reader and help students gain an overall understanding of the topic.

The section on ‘system development’ seems to be well-researched, however the formatting of the content in short, one-sentence dot points makes it difficult to read and incongruent, so writing this out in small paragraphs would improve the readability. The capitalization of some words is unnecessary in both the dot points and the table, creating inconsistencies in the formatting. Also, some words are unnecessarily bolded which detracts from the aesthetic appeal of the page. However, the inclusion of a table to summarise the timeline information is an effective tool, although there is much more information provided for the male system than female system. It is really good to see the use of an image as it is relevant and clearly compares the male and female system development side-by side. I also think the video inclusion is fantastic as it would be an effective way to learn for a reader with no previous knowledge, making the page more interactive.

The section on ‘current research, models and findings’ contains lots of relevant information, however this is not referenced in-text and it is thus unclear where the information has been derived from. There also seems to be some unevenness between the depth of information between male and female systems, which some more research can easily remedy. In terms of current findings, the listing of the information in dot points makes it easier to read, however there are some parts italicised and capitalized that are not needed. It is great to see some hand-drawn images as these are simplistic, colourful and effective ways to accompany the text, adding to the page’s appeal. Take care to properly include images, as one of them appears as ‘alt text’ and the link does not show the image itself. Although some references appear under a ‘references’ title in this section others appear as a website links; formatting of these could improve neatness.

The following section on ‘historic findings’ contains evidence of extensive research as it is very detailed and well-written. However, I would consider breaking this part up into smaller sections using dot points as large paragraphs seem tedious to read. The hand-drawn image is a good inclusion, but labelling of it would be effective and adding a couple more would break up the long section visually. Also, there seem to only be in-text citations after long chunks of information; perhaps more sources should be used/consulted.

Lastly, the abnormalities section was comprehensive and detailed and enough information was given on some examples. This was just the right amount of content, as any more would seem excessive. Adding some more images with appropriate captioning is advised also. I liked that the references were listed altogether at the end of the page, making it neat and tidy. Overall, a solid project which just needs some formatting to improve further.

Group 5- Integumentary

The introduction of this page was good as it provided a great overview and insight into what the project would later go on to discuss. Perhaps a little information on defining the integumentary system itself would be valuable though, to let the reader know the constituents.

The sub-sectioning of the page’s content into ‘introduction, ‘development overview’, ‘recent findings’, ‘historic findings’ and ‘abnormalities’ was clever, as the development section then went on to describe each component such as skin, nails etc. This effectively segmented the information into smaller chunks that could easily be navigated to. I especially liked the use of the table in the ‘skin’ section, showing the week of development, description and image corresponding alongside it, as it provided a holistic approach to that section. However, there were no in-text citations in the ‘skin’, ‘hair’, ‘nail’ or ‘teeth’ sections, hence the source of the information is unclear. To do this correctly, Dr Hill’s Wiki help page should be consulted.

The use of various images with labelled captions was a strength of this project, as the pictures were effective in balancing the text components to make the page more visually appealing. Most of the sections under ‘development overview’ have a decent amount of content, however the ‘nails’ part is a bit lacking and also needs to be correctly cited. The use of the table of images alongside the description of developmental stage in the ‘teeth’ section was very good to include, however the image is captioned ‘The stages of embryonic teeth development’. This may be irrelevant as the project’s focus is on fetal development. Further research into this area may uncover more relevant information.

Although the section on ‘recent findings’ contains a lot of information, the use of chunky paragraphs detracts from the readability of the page and the purple boxes could be summarised into dot points to help the reader understand the content more quickly. The section on historic findings seems concise and relevant, however the image included says it has been removed/deleted, so this requires editing. Lastly, the ‘abnormalities’ section was very well-structured and written, as each example had a captioned image accompanying it to help the reader visualise. This section was also well-done in terms of in-text citations, with an extensive reference list provided at the end. Perhaps consider adding some student-drawn images and possibly a relevant video, but otherwise it is very good as it is. Overall, the project has a good layout and a decent amount of content; with some editing and formatting it can be improved further.

Group 7- Neural

Overall, the project contains a decent amount of content as it is, split up into appropriate subheadings, considering the large scope of the nervous system. The introduction provides a succinct description of the CNS, however including an outline of what topics the page intends to cover would be good to orient a reader that approaches the page for the first time. The descriptions of the brain and spinal cord are well-written, however require in-text citations and some words need not be capitalised e.g midbrain, hypothalamus. These can be easily fixed with proof-reading and further editing.

The use of an image to illustrate fetal development was a very good idea; although the diagram is itself seems complicated, it can be explained well with the accompanying text beneath. The segmentation of the timeline into 4 different parts made it easier to follow, although I would consider placing the images on the right hand side of the page to reduce the vertical length of the page and the scrolling required to navigate through it. The section on ‘brain development’ contained some relevant information, formatted in dot points which improves readability, however in-text citations are needed to allow the reader to source the information if required. The use of the table in this part was effective also; it was concise and straight to the point.

Although the brain development section was very well detailed, there was no information in the spinal cord and meninges sections; with further research, these need to be evened out, along with the current findings section which is just references at this stage, but still a good start to finding sources of information. The section on current research is well-detailed and contains a lot of information too, which is good to see. Fiinally, I thought the section on abnormalities was very well done, using subheadings to segment the content. The use of dot points allowed the information to be easily read off the page, and use of images to accompany them helped the reader to visualise the conditions. There is one image however that has been incorrectly uploaded; consulting Dr Hill’s Wiki help page can remedy this. Also, the placement of all references under one list at the end of the page was effective in neatening it up, that was very well done too.

Overall, the project’s content has been well-researched and there is evidence of good teamwork and communication. Perhaps the use of some student-drawn images and inclusion of a relevant video may be areas of improvement.

Group 8- Musculoskeletal

Overall, the project has some very detailed sections and some sections where content is scarce. It would be helpful to start off with an introduction of the musculoskeletal system so the reader is aware of its components and what the page intends to cover. The timeline of muscle development has good potential, I understand it is still being planned at this stage and with further research, it could definitely be effective. A table format would be useful to present this information. The following sections on background embryonic development and fetal myogenesis are well-researched and have a lot of content, however I would consider breaking it down into dot points to improve readability. The sections are cited correctly in-text though, which is good to see.

There is much more improvement in the tendon and second trimester development sections, as the chunks of text have been reduced to provide a succinct summary, however these need to have citations also. The use of some images here, either hand-drawn or from online would be beneficial, to have a balance between text and pictures and make the page more visually appealing. Other than the abnormalities section which provides a good, concise summary of Duchenne Muscular Dystrophy, the following sections seem to be only references at this stage. As long as these are used to compose some relevant paragraphs/dot points, this is fine considering there is still time to improve the page.

Overall, this page has good potential as the groundwork has been completed; it is now more a matter of writing up more information, adding images and possibly a relevant video. The part on ‘making gains’ would need to be removed for the final, but otherwise, it is definitely a decent amount of work so far, especially considering the few group members involved.

--Mark Hill You have provided a comprehensive review of the projects giving a balanced critical assessment of the content. Hopefully your peers will take notice of your recommendations. (10/10)

Lab 10

Research article- Stage-dependent requirement of neuroretinal Pax6 for lens and retina development

Methods summary

Embryonic mouse stem cells from timed pregnant females were recombined in a homologous fashion to form floxed Pax-6 alleles in the experimental mice. These embryos were then harvested, fixed, PBS-washed, cryopreserved and sectioned for immunohistochemistry staining in which embryos were permeabilized and washed with PBT and incubated. This was followed by RNA in-situ hybridisation, where RNA polymerase was used to create antisense mRNA probes, then X-gal staining occurred.

The study then measured the length of the cell cycle phases of the mice embryos to assess the role of Pax6. Using wild-type littermates as controls, they found the proportion of proliferating retinal progenitor cells (RPCs) by injecting timed pregnant females with BrDU, then fixing, cryopreserving and sectioning them. Following this was a process of antigen retrieval with several incubations to calculate cell proliferation rate.

Finally, the cell cycle rate at embryonic days 11.5 and 13 were found and the cell cycle and S phase lengths were determined, also calculating the total length of the G1, G2 and M phases. These results were then quantified by statistical analysis using a t-test, counting 3 fields for every individual eye.

Results summary

By combining the findings of the separate sections of the experiment, the study concluded that Pax6 has an essential role in both lens and retinal cell formation. It was found that Pax6 deletion strong hypocellularity in early RPCs. There was high expression of Pax6 in the surface ectoderm (SE) and optic vesicle (OV) control embryos while the levels of Pax6 stayed constant and unchanged even after Pax6 elimination in the OV, lens pit and SE. The hypocellularity of the retina was more pronounced with time, and retinae deficient in Pax6 became smaller progressively.

Another finding was that the population size of the RPCs was reduced by changed cell cycle length and imbalance between cells exiting the cell cycle and proliferation of eye progenitor cells. It was again observed that retinal cells deficient in Pax6 were very hypocellular and cycling cells were centrally localised in the retina. An increased rate of cell death was observed with increasing time and total cell cycle length of Pax6-deficient RPCs was significantly longer than the control embryos. Also, the down-regulation of cyclin D1 in Pax6-deficient mice suggested that absence of Pax6 drives RPCs to cell cycle exit. Together, these findings suggested that positive progression of RPCs through the cell cycle is regulated by Pax6.

Finally, another conclusive result reached by this study was that the absence of Pax6 in RPCs causes a hindrance in their proper differentiation program into retinal cells; indicating the essential role of Pax6 in proliferation of early progenitors. It was also found that deficiency of Pax6 in optic vesicles could lead to the arrest of lens development during the interaction of the OV and SE. As neither the lens nor retina was formed properly in Pax6 deficient embryos, this indicated the importance of Pax6 presence in the optic vesicle for correct eye morphogenesis in the developing embryo.

CMs and vascular cells were simultaneously induced using a differentiation protocol wherein the levels of gene expression of cardiac mesoderm and progenitor genes peaked at day 5 of differentiation. They found that mesoderm-to-CM differentiation enhancement occurred during days 5-7 due to Dkk1 addition (an antagonist). When vascular cell induction with CMs using VEGF was attempted instead of Dkk1, this led to induction of ECs and CMs together. The study found that the cell populations had a composition of vascular endothelial cadherin, platelet-derived growth factor receptor beta and positive MCs. These findings collectively suggested that a change from ‘only CMs’ to ‘CMS and vascular cells’ could be induced by selectively controlling the direction of differentiation of cardiovascular cells.

The experiment also tried to develop sheets of cardiovascular cells from hiPSCs by continuous culture, re-plating and incubations. It was found that reducing temperature served to re-assemble self-pulsating sheets of cells after heating and that sheets were composed of 3-4 layers supported by collagen fibres in a stratified manner. These sheets were shown to have CMs evenly distributed throughout them via immunohistochemical techniques. Within the cell sheets were; CMs, MCs and undifferentiated cells and this composition was attributed to possible apoptotic cell death reducing numbers and reduced proliferation efficiency. Together, these results suggested that it was the hiPSCs only that had generated the CMs and vascular cells, forming a structure that resembled cardiac tissue very realistically.

Lastly, Masumoto et al also found that the transplantation of hiPSCs with CTSs could alleviate dysfunctions in the cardiovascular system even after infarction in rats, as all rats had survived the period of post-transplantation without signs of tumours arising. Using echocardiogram technology, it was observed that anterior wall contraction had been brought back to normal, left ventricle systolic function had improved and that there had been reduced thickening of the wall of myocardial infarcts. Furthermore, staining with Sirius red found that the rate and extent of fibrosis was significantly reduced after hiPSC-CTS transplantation.

In conclusion, Masumoto et al concluded that there remained ample scope for hiPSCs to regenerate cardiac tissue and thus restore cardiac function, if their capabilities were studied more widely by other researchers. The replacement ability and biomedical potential of these pluripotent stem cells hold great promise for the future of stem cell therapy.